Tryptic Peptide Signal Enhancement via MALDI IMS Post-ionization (MALDI-2) from Thin Tissue Sections

24 December 2020, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the highly multiplexed, unlabeled mapping of analytes from thin tissue sections but further work is needed to improve sensitivity and depth of coverage for protein and peptide IMS. Laser-based post-ionization (MALDI-2) has been shown to increase sensitivity for numerous molecular classes for MALDI but this has not been demonstrated for peptides. Here, we demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney with MALDI-2. Proteins were digested in situ using trypsin prior to the IMS analysis with MALDI (here, MALDI-1) and MALDI-2. For identification of peptides and proteins from MALDI IMS, a tissue homogenate was analyzed via LC-MS/MS for bottom-up proteomics and the proteins identified via LC-MS/MS were further ‘digested’ in silico to generate a database of theoretical peptides to match to MALDI IMS data sets. Peptides were tentatively identified by matching the MALDI peak list to the database within 5 ppm error that resulted in 170 ± 37 peptides and 885 ± 73 peptides for MALDI-1 and MALDI-2, respectively. Protein identifications were similarly made by linking IMS data to LC-MS/MS results wherein positive identifications required two or more peptides to be detected per associated protein. This resulted in 55 ± 13 proteins identified with MALDI-1 and 205 ± 10 with MALDI-2. MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and it greatly increases the number of proteins identified during IMS experiments.


imaging mass spectrometry
matrix assisted laser desorption/ionization mass spectrometry
imaging mass spectrometry techniques
peptide imaging
protein imaging mass spectrometry
Spatial proteomics

Supplementary materials

Supplementary Figures Peptide IMS MALDI-2 McMillen-et-al


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